This invention relates to assays for ligands, e.g., antigens, in a liquid sample such as a body fluid. More particularly, the invention relates to a method and apparatus for the detection of a ligand in a body fluid such as urine using a conjugate comprising colored particles and a novel flow-through test cell.
Many types of ligand-receptor assays have been used to detect the presence of various substances, often generally called ligands, in body fluids such as urine. These assays involve antigen antibody reactions, synthetic conjugates comprising radioactive, enzymatic, fluorescent, or visually observable metal sol tags, and specially designed reactor chambers. In all these assays, there is a receptor, e.g., an antibody, which is specific for the selected ligand or antigen, and a means for detecting the presence, and often the amount, of the ligand-receptor reaction product. Most current tests are designed to make a quantitative determination, but in many circumstances all that is required is a positive/negative indication. Examples of such qualitative assays include blood typing and most types of urinalysis. For these tests, visually observable indicia such as the presence of agglutination or a color change are preferred.
Even the positive/negative assays must be very sensitive because of the often small concentration of the ligand of interest in the test fluid. False positives can also be troublesome, particularly with agglutination and other rapid detection methods such as dipstick and color change tests. Because of these problems, sandwich assays and other sensitive detection methods which use metal sols or other types of colored particles have been developed. These techniques have not solved all of the problems encountered in these rapid detection methods.
It is an object of this invention to provide a rapid, sensitive method for detecting ligands in body fluids. Another object is to provide an assay which has high sensitivity and fewer false positives than conventional assays. A further object is to provide a test cell for detection of low levels of ligands in body fluids. Another object is to provide an assay system which involves a minimal number of procedural steps, and yields reliable results even when used by untrained persons.
These and other objects and features of the invention will be apparent from the following description, drawings, and claims.
The invention features a method and test cell for the detection of a preselected ligand in a liquid sample such as a body fluid.
The test cell useful in the practice of the invention has an elongate outer casing which houses an interior permeable material, e.g., glass fiber, capable of transporting an aqueous solution by capillary action, wicking, or simple wetting. The casing defines a sample inlet, and interior regions which, for ease of description, can be designated as a test volume and a reservoir volume. The reservoir volume is disposed in a section of the test cell spaced apart from the inlet, and preferably is filled with sorbent material. The reservoir acts to receive liquid transported along a flow path defined by the permeable material and extending from the inlet and through the test volume. In the test volume is a test site comprising a first protein having a binding site specific to a first epitope of the ligand immobilized in fluid communication with the flow path, e.g., bound to the permeable material or to latex particles entrapped in or bonded to the permeable material. A window such as a hole or transparent section of the casing permits observations of the test site through the casing wall.
In a preferred embodiment, the flow path is restricted or narrowed in the test area, thereby channeling and concentrating fluid flow into contact with the test site. It is also preferred that the test cell include a solution filtering means disposed in the flow path between the sample inlet and the test site. The filtration means can comprise a separate, conventional filter element disposed within the casing of the test cell in fluid communication with the permeable material defining the flow path, but preferably is defined simply by a portion of the permeable material itself. The provision of such a filtration means in the test cell has the effect of removing by entrapment from impure samples, such as urine samples, a portion of the particulates and nonspecific interfering factors which sometimes cause false positive readings.
The method of the invention requires the use of a conjugate comprising a second protein bound to colored particles such as a metal sol or colloid, preferably gold. The conjugate can take two distinct forms, depending on whether the assay is designed to exploit the xe2x80x9csandwichxe2x80x9d or xe2x80x9ccompetitivexe2x80x9d technique.
In the case of the sandwich technique, the second protein comprises a site which binds to a second epitope on the ligand. This type of conjugate reacts with the ligand to form a complex in the liquid sample. The complex is detected by visual observation of color development at the test site in the test cell. At the test site, the ligand bound with the conjugate reacts with the immobilized first binding protein to form a xe2x80x9csandwichxe2x80x9d of the first protein, ligand, second protein, and colored particles. This sandwich complex is progressively produced at the test site as sample continuously passes by, filling the reservoir. As more and more conjugate is immobilized, the colored particles aggregate at the test site and become visible through the window, indicating the presence of ligand in the liquid sample.
In the case of the competitive technique, the second protein binds with the first protein in competition with the ligand. The second protein comprises, for example, an authentic sample of the ligand or a fraction thereof which has comparable affinity for the first protein. As the liquid sample is transported in contact with the test site, ligand, if any, and the conjugate compete for sites of attachment to the first protein. If no ligand is present, colored particles aggregate at the test site, and the presence of color indicate the absence of detectable levels of ligand in the sample. If ligand is present, the amount of conjugate which binds at the test site is reduced, and no color, or a paler color, develops.
In one embodiment of the invention, the test liquid is mixed with the conjugate outside the test cell. In another embodiment, the conjugate is disposed in freeze-dried or other preserved form on the permeable material between the inlet and the test site, and the sample liquid resolubilizes the conjugate as it passes along the flow path.
Color development at the test site may be compared with the color of one or more standards or internal controls to determine whether the development of color is a true indication of the presence or absence of the ligand, or an artifact caused by nonspecific sorption.
In one embodiment employing the sandwich technique, the standard consists of a negative control site, preferably disposed adjacent the test site, and visible through a second window proximate the first. The negative control site preferably is prepared identically to the test site, except immobilization of the first binding protein is omitted. Therefore, although the conjugate will reach the control site, it aggregates due only to non-specific binding. If the test site is not appreciably more intense in color than the control site, the assay is considered negative.
In another embodiment, the assay and test cell may include a positive control. Thus, when exploiting the sandwich technique, the cell may have an authentic sample of the ligand immobilized at a control site. If no color develops at this control site, the assay is considered inconclusive. When exploiting the competitive technique, the development of color at the positive control site means the assay results are inconclusive.
Broadly, the method of the invention involves the use of a test cell of the type described above to achieve an easily readable, sensitive, reproducible indication of the presence of a ligand, e.g., human chorionic gonadotropin (hCG), in a test sample such as a human urine sample. The method involves the step of transporting the sample and a conjugate comprising a protein bound to a metal sol or other colored particle along a flow path and in contact with a test site comprising immobilized binding protein specific to an epitope of the ligand, and preferably also in contact with a control site. Preferably, the colored particle comprises a gold sol; the flow path in the region of the test site is reduced in cross-section relative to other parts of the flow path; the sample is passed through a filtration means after it enters the test cell but before it contacts the test site; and the test site comprises latex particles entrapped or otherwise fixed in the flow path having the immobilized protein on their surface. In the practice of the process, either the conjugate is premixed with the sample, or the conjugate is disposed in preserved form, e.g., lyophilized, in the flow path between the inlet and the test site. In either case, placement of the test cell in the sample, or application of the sample to the inlet, initiates flow, and the result is read by observing color development as the test site, or by comparing the color of the test site and control site.
The use of the colored particle detection system in combination with the filtration means, the concentrating effect of flow of the sample, and the ease of comparison between the colors of the test and control sites, together enable construction of a family of extremely sensitive assay systems which minimize false positives and can be used effectively by untrained persons.